Principal Investigator Biotechnology, Integral Membrane Proteins (IMP)
Screening and Production of Human Proteins Using Baculovirus and BacMam
The SGC promotes research advancement through our open access policy. Globally, we have solved more than 2000 human protein structures and 13 novel integral membrane proteins (IMPs). Our well established high-throughput processes for production and validation of intracellular and membrane proteins will be presented along with their success rates. In addition, optimization strategies employed over the past 15 years to tackle the most recalcitrant proteins, including mutagenesis, mammalian (BacMam) expression, fluorescence-detection size exclusion chromatography (FSEC), and twin-step purification will be discussed.
Nicola Burgess-Brown is the Principal Investigator of the Biotechnology Group at the SGC, responsible for managing all biotech research for the Oxford site. Nicola’s team develops high-throughput screening processes from cloning to expression, purification, and mass spectrometry analysis of human proteins for structural and functional studies. Nicola obtained a first class degree in Applied Biochemical Sciences from the University of Ulster in 1997, then worked as a molecular biologist for SmithKline Beecham. She received her Ph.D. in Molecular Microbiology at the University of Nottingham in 2001 and then moved back to industry to work on high-throughput cloning and validation of therapeutic cancer antigens for Oxford Glycosciences and subsequently Celltech R&D.
Developing a Potency Assay for a Proprietary AAV-Based Genome Editing Platform Technology
The GeneRide platform utilizes the natural process of homologous recombination to achieve targeted genome editing without the use of exogenous nucleases or promoters. Developing a potency assay for this unique technology poses additional challenges over those for canonical gene therapy products, including the requirement of a highly sensitive detection method to measure low levels of genome integration. The first GeneRide candidate, LB-001, is currently under clinical development for the treatment of methylmalonic acidemia. LB-001 targets site-specific integration into the albumin locus to allow the gene of interest (MMUT) to be expressed concomitantly with albumin. In order to control LB-001 product activity and assess lot-to-lot consistency, a cell-based assay was developed to measure fused mRNA expression as a surrogate of biological activity. The assay was developed in a cell line that naturally expresses albumin and can thus drive expression of the MMUT gene upon site-specific integration. Fused mRNA is quantified using primers overlapping the host genome and the transgene DNA to ensure that only the integrated product is detected. The results show that AAV-driven homologous recombination is reproducible in vitro, which allows for the qualification of assay control material. The method was tested for linearity, repeatability, and specificity. Examination of the assay data demonstrates that this method is suitable for assessing the relative potency of integrating GeneRide vectors.
Matthias Hebben has been serving as VP Technology Development at LogicBio Therapeutics since February 2019. Before that, he served as Director of AAV Technology Development and Head of Bioprocess Development at Genethon for six years where he managed the design and scale-up of manufacturing processes for AAV and LV vectors. Prior to his role at Genethon, Matthias was Director of the Virology Unit at Vivalis for four years. Before that, he occupied several roles in the animal health industry at Intervet Schering Plough and Virbac between 1999 and 2008. Matthias has a PhD in molecular biology from the University of Nice Sophia Antipolis (France) and a bioprocess engineer degree from the University of Strasbourg (France).
Biophysicist Research Scientist, Molecular Biophysics and Integrated Bioimaging, Structural Biology
New Technologies for Robust and Efficient Large-Scale Production of Recombinant Multi-Protein Complexes
MacroBac is a multigene baculovirus system that uses ligation-independent cloning for efficient cloning and assembly that is equally well-suited for either single or high-throughput cloning reactions. MacroBac vectors are polypromoter to minimize gene order expression level effects seen in many polycistronic assemblies. Large assemblies are robustly achievable, and we have observed significant increases in expression levels and quality of large, multiprotein complexes over traditional coinfection with multiple, single-gene baculoviruses.
Jill O. Fuss, PhD is a Research Scientist at Lawrence Berkeley National Laboratory and Founder and Chief Technology Officer of Cinder Biological, Inc. Dr. Fuss earned a BA in environmental science from Wesleyan University, and a PhD in molecular and cell biology from the University of California, Berkeley. Her postdoctoral fellowship was performed at Lawrence Berkeley National Laboratory (LBNL) where she received a National Institutes of Health National Research Service Award and was named a US Department of Energy Outstanding Mentor. She received two LBNL Director’s Awards for Exceptional Achievement in 2013 and 2016 and was recognized as a Berkeley Visionary in 2015.
Baculovirus-Cell Interactions in the Insect Midgut: Transcriptome Profiling and Envelope Protein Trafficking
In nature, the midgut is the first tissue encountered during baculovirus infection of the animal. The virus must navigate through the midgut cells and bud into the hemocoel to achieve successful systemic infection. To better understand the complex interactions between virus and host, we have used transcriptomic approaches to examine both viral and host transcription profiles in the midgut (the primary phase of infection) in comparison with similar studies in cultured insect cells (which represent the secondary phase of infection). Also, to better understand how many viruses navigate through the midgut, we have recently developed several new experimental systems for broad screens of host cell proteins and pathways involved in viral envelope protein trafficking in cultured insect cells and in the insect midgut.
Gary Blissard is a Professor at the Boyce Thompson Institute at Cornell University, and an Adjunct Professor in the Departments of Microbiology and Immunology, as well as Entomology, at Cornell University. His studies have focused on the structure, function, and trafficking of viral envelope proteins, viral entry and egress, and viral and host cell gene expression.
Professor of Virology, Department of Pathogen Molecular Biology
Use of Recombinant Proteins and Particles for Molecular Studies and Novel Vaccines
Large-scale interactome maps of eukaryotic systems have found that biologically active proteins are often part of large multi-subunit complexes. Therefore in order to properly study protein activities in vitro, or for vaccine purposes, it is essential that all the subunits of large protein complexes are functional and correctly assembled. Baculovirus expression is an excellent system for the generation of multi-subunit protein complexes and we have been successful in recovering enzymatically active complexes for a variety of proteins. The protein complexes that we have tested range from relatively simple enzymatic systems with small numbers of subunits to large multi-layered complexes and other large multi-subunit particulate structures. In particular, we used an architecturally complex model virus with a view to understanding the role of each viral protein in the virus replication cycle. The model virus is composed of seven discrete proteins in a specific but non-equimolar ratio that are organised into four layers of two shells, the inner core and outer capsid and enclosed genome of 10 double-stranded (ds) RNA segments. Bluetongue virus (BTV) is an insect vectored emerging pathogen of wild ruminants and livestock, causing disease in sheep, goats, and cattle with mortality reaching 70% in some breeds of sheep, with high economic consequences.
We expressed each viral protein, then purified and analysed the role of each in the virus life cycle. Data obtained from these early studies have defined the key players in BTV entry, replication, assembly, and egress. Specifically, it has been possible to determine the complex nature of the virion through three-dimensional structure reconstructions; atomic structure of proteins and the internal capsid; the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell and the protein sequestration required for it; and the role of three nucleoid structuring (NS) proteins in virus replication, assembly, and release. Based on information gained from structural and molecular studies, a novel technology has been developed to produce highly efficacious safe vaccines for BTV and related viral diseases.
We undertook a series of vaccination trials in animals. We found that the recombinant receptor binding protein VP2, alone or in combination with membrane penetration protein VP5, could afford protection against virulent virus challenges. This led to the observation that presenting VP2 in the appropriate conformation would reduce the amount of protein required for vaccination. This was taken to a logical conclusion and the two BTV outer capsid proteins together with scaffolding core proteins, VP3 and VP7, were expressed by a recombinant baculovirus expression system to generate double-capsid virus-like particles (VLPs). The BTV VLPs, the first in the field (1990), had the characteristic icosahedral structure, the most complex protein assembly produced, with the four proteins in non-equimolar ratios which assemble into a particle with a total of 1440 subunits. This particle acts to mimic the overall structure and immunogenicity of authentic virus and efficiently stimulates both B-cell and T-cell responses, conferring protection against virulent virus challenge. Further, it was possible to co-express the outer capsid proteins from different serotypes onto the conserved inner core and these heterologous particles, as expected, were highly protective against virulent virus challenges in sheep and generated multiple vaccine strains for BTV. The same technology was then adopted for related viruses and also for many other viruses that are currently commercialized.
Professor Polly Roy began her education in India, where she won a fellowship to New York University for her PhD under the supervision of the renowned molecular biologist, Sol Spiegleman. She continued with a three-year postdoctoral position in virology at the Waksman Institute, Rutgers University and then joined the University of Alabama, Birmingham. There she established her own virology research group, becoming a full Professor in 1986.
In 1987, she received a senior International Fogarty fellowship to study at the University of Oxford where she established a UK-based research group. In 2001, Professor Roy took the Chair of Virology at the London School of Hygiene and Tropical Medicine, where she continues to lead a large research group working on basic virology and applied vaccine research. Her work focuses on the molecular biology of RNA viruses, using Bluetongue virus (related to human rotavirus) as a model. These studies have contributing to many areas of virology such as virus structure and assembly, RNA replication, and novel vaccine development. Professor Roy was the first to demonstrate the assembly of virus-like particles (VLPs), a technology which has since been applied to many other viruses including successful vaccine development for human papillomavirus (HPV), influenza, and SARS. Her research group still continues to use the baculovirus expression system for both basic research and application. She recently pioneered the synthesis of infectious viruses solely from synthetic genes, leading the way for the development of yet newer vaccines and therapies.
Professor Roy has supervised over 150 postdoctoral and post-graduate researchers and published over 350 papers in various high impact journals, as well as a number of chapters in medical, veterinary, and virology books including Field’s Virology and various encyclopaedias. She is also an editor of several books. Her research has been funded by many agencies including the National Institutes of Health (NIH), National Science Foundation (NSF), United States Department of Agriculture (USDA), Biotechnology and Biological Sciences Research Council (BBSRC), Medical Research Council (MRC) and the Wellcome Trust, UK, as well as several European Commission (EC) grants.
Professor Roy has served on various international scientific organizations, committees, and boards. She has organized several highly successful international conferences, particularly on the subject of virus structure and assembly, double-stranded (ds) RNA viruses, and several viral vaccine symposia.
In 2006 Professor Roy was elected a Fellow of the Academy of Medical Sciences, UK and in 2012 she received the Gold Medal for her contribution to science and technology from the then Indian Prime Minister Manmohan Singh. She is one of the three BBSRC Scientific Innovators of the year and in 2014 received the “Order of the British Empire” for her achievements in Virus Research.
Considerations for the Development of Gene-Edited Stem Cell Therapies
The era of gene editing has unlocked the potential to cure diseases that previously had few treatment options. In particular, gene-edited stem cell therapies have shown great promise in the treatment of β thalassemia and sickle cell disease, and also offer the opportunity to change the treatment landscape in hematological malignancies. This talk will discuss some of the points to consider when designing and developing a gene-edited stem cell manufacturing process.
Brent Morse is the Head of Process and Analytical Development at Vor Biopharma. Prior to Vor, he served as Director of Analytical Development at CRISPR Therapeutics where he established the Analytical Development group and supported multiple regulatory filings for gene-modified stem cell and CAR T-cell therapies. Before that, he was Director of Bioanalytical Development at EPIRUS Biopharma where he developed biosimilarity protocols for multiple products. Brent has also held several R&D positions within the biotech and pharmaceutical industry, including roles at Abbvie and Adnexus (Bristol-Myers Squibb).
Research Specialist - Baculovirus-Insect Cell Technology, UQ Protein Expression Facility
Learning from Hundreds: A Data-Driven Approach to Harness the Power of Baculovirus-Insect Cell Technology
The baculovirus-insect cell expression system is a widely used eukaryotic expression system for heterologous protein production with growing industry and research applications. The recent technology developments and the availability of commercial baculovirus expression kits have eased the use of this highly complex expression platform. However, to achieve milligram quantities of pure homogenous proteins, optimization of production parameters for each protein is required. Data obtained from hundreds of expression screens conducted on both intracellularly- and extracellularly-expressed proteins have provided insights on the critical factors affecting protein expression. The choice of insect cell line, culture temperature, secretion signal peptide, and baculovirus expression system have crucial consequences on the heterologous expression of proteins. In this presentation, we will discuss the trends observed from screening multiple parameters and the effect of their interactions.
Mercè Salla-Martret is the Unit Head of Baculovirus/Insect Cell Expression technologies at the Protein Expression Facility (PEF) at the University of Queensland. PEF is Australia’s leading protein research facility that specialises in recombinant protein production for academic and industry researchers. Mercè obtained her Bachelor of Science in 2005 at the University of Barcelona (UB, Spain) and then a PhD in Biochemistry and Molecular Biotechnology at the Spanish National Research Council (Consejo Superior de Investigaciones Científicas, CSIC) and at the Center for Research in Agricultural Genomics (CRAG) in 2012. After two years of postdoctoral research on protein-protein interactions, she relocated to Australia and recently joined PEF in 2019 to work on the implementation of baculovirus expression technologies for high-throughput screening and large-scale protein production.
Regents Distinguished Professor, Diagnostic Medicine Pathobiology & KBA Eminent Scholar, College of Veterinary Medicine
Baculovirus-Based Vaccines for Rift Valley Fever Virus
Rift Valley fever (RVF) is a zoonotic disease that causes severe epizootics in ruminants, characterized by mass abortion and high mortality rates in younger animals. The development of a reliable challenge model in target animals is an important prerequisite for evaluation of existing and novel vaccines. We conducted studies aimed at comparing the pathogenesis of RVF virus (RVFV) infection in US sheep and cattle using two genetically different wild-type RVFV strains, SA01-1322 and Kenya-128B-15. The Kenya-128B-15 strain manifested higher virulence compared to SA01-1322 by inducing more severe liver damage, and longer and higher viremia in both sheep and cattle. Genome sequence analysis of both isolates revealed sequence variations between the two isolates, which potentially could account for the observed phenotypic differences. These results demonstrate the establishment of virulent target host challenge models for vaccine evaluation based on the RVFV strain Kenya-128B-15. There is currently no fully licensed RVFV vaccine suitable for use in livestock or humans outside endemic areas. Therefore, we evaluated the efficacy of a recombinant subunit vaccine based on the RVFV Gn and Gc glycoproteins. The vaccine elicited strong virus neutralizing antibody responses in sheep and cattle and is DIVA (differentiating infected from vaccinated animals) compatible. Furthermore, a group of sheep was vaccinated subcutaneously with the Gn/Gc-based subunit vaccine candidate, and then challenged with the virulent Kenya-128B-15 RVFV strain. The vaccine elicited high virus neutralizing antibody titers and conferred complete protection in all vaccinated sheep, as evidenced by prevention of viremia, fever, and absence of RVFV-associated histopathological lesions. We conclude that the subunit Gn/Gc vaccine represents a promising strategy for the prevention and control of RVFV infections in susceptible hosts.
Dr. Richt came to Kansas State University in 2008 as the Regents Distinguished Professor and Kansas Bioscience Eminent Scholar. In 2010, he became Director of the Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD). He received his doctorate in veterinary medicine (DVM) from the University of Munich and his PhD in Virology and Immunology from the University of Giessen, both in Germany. After coming to the United States in 1989, he completed three years of postdoctoral/residency studies at the Johns Hopkins University in Baltimore, Maryland, and later served for eight years as a Veterinary Medical Officer at the National Animal Disease Center (USDA-ARS) in Ames, Iowa. He has edited several books, published more than 240 peer-reviewed manuscripts, and raised more than $40 million in grants for veterinary research since 2008.
Dr. Richt is a pioneer in veterinary science, most notably in the “One Health” field. His work on high consequence pathogens with zoonotic and transboundary potential led to strategies to identify, control, and/or eradicate such agents. His basic and applied research includes studies on animal influenza viruses, animal prion diseases including bovine spongiform encephalopathy (BSE), Rift Valley fever virus (RVFV), Schmallenberg virus (SBV), African swine fever virus (ASFV), vesicular stomatitis virus (VSV), and Borna disease virus (BDV). Dr. Richt established the first reverse genetics system for swine influenza viruses (SIV), made seminal contributions to the development of a modified live SIV vaccine (sold in the US as Ingelvac Provenza™), and to understanding the virulence of the reconstructed 1918 “Spanish Flu” virus in livestock. He identified an atypical BSE case with a causative mutation (“genetic BSE”), used gene-editing approaches to develop the first prion protein knock-out cattle which are resistant to prion infection, and provided valuable information on the host range of animal prions essential for risk analysis. Dr. Richt’s RVFV work led to the development of novel domestic and wild ruminant models for RVF and a safe, efficacious, and DIVA-compatible subunit vaccine. For ASFV, he is developing subunit and modified live virus vaccine candidates as well as point-of-need diagnostic tools to protect swine from this devastating disease. As founding Director of a Department of Homeland Security (DHS) Center of Excellence, he is supporting DHS and USDA in protecting US agricultural systems and public health from devastating animal and human diseases.
Optimizing Cell Manufacturing Processes for the Treatment of Severe Genetic Diseases and Cancer
The cell and gene therapy space has seen a great deal of growth with an increasing number of ongoing and newly-initiated clinical trials for different disease indications. The speed at which these cell therapies move through the clinic and the advancements in manufacturing technology have brought forth both unique challenges and opportunities in the last decade. Two case studies will be presented to demonstrate some of the challenges with developing a robust process for commercial manufacturing of cellular therapies. The first case study examines the relationship between the different inputs in the transduction operation for hematopoietic stem cells and identifying some of the critical outputs which determine clinical success. The second case study investigates the different media formulations necessary for T-cell expansion to manufacture the clinical doses required for CAR T-cell or TCR-based therapies. Both case studies will explore how a process development team develops and optimizes an autologous cell manufacturing process, which helps gain better process understanding overall.
Liz is currently the Associate Director of Cellular Process Development at bluebird bio managing the clinical programs in severe genetic diseases (SGD) and oncology from preclinical to commercial. Before bluebird bio, Liz started her industry career at Novartis Pharmaceuticals in New Jersey working on the development of Kymriah as one of the first approved CAR T-cell products for CD19. Liz was part of the analytical and process science team involved in process characterization and development for biologics license applications (BLAs), supplemental BLAs (sBLAs), and marketing authorization applications (MAAs). She was then promoted to process lead at Novartis for their pipeline projects and worked on the investigational new drug (IND) application filing for the next-generation CAR T-cells. Liz received her PhD in Biochemistry at the University of Illinois and then did her postdoctoral work at Duke University under the guidance of Dr. Bruce Sullenger and Dr. Smita Nair.
Routine Size, Mass, Aggregate Level, Total Concentration and Empty:Full Ratio Determinations for AAV Particles by SEC-MALS
Mass and size data can be advantageous in assessing the overall quality of adeno-associated virus (AAV) materials. Recent improvements in multi-angle light scattering (MALS) data analysis methods suggest SEC-MALS can be applied to estimate both the total capsid titer and the extent of DNA encapsulation (i.e. empty:full ratio) for well-behaved AAV samples. We developed and tested a SEC-MALS approach using AAV "empties," "fulls," and mixtures of the two samples obtained from a single batch of cesium chloride (CsCl) gradient-purified AAV. After refining our acquisition and data processing methods, we found the empty:full ratio determined by SEC-MALS comparable to that obtained from both non-staining cryo-transmission electron microscopy as well as analytical ultra-centrifugation (AUC). We also found the total capsid titer determined by SEC-MALS for these samples to be consistent with the total titer back-calculated from AUC data and vector genome concentration (i.e. active titer). Preliminary tests using SEC-MALS for empty:full ratio show the method is fast, robust, reproducible, and executable across a useful concentration range for AAVs. The method neither performs nor requires separation of empty from full AAV species, meaning little to no adaptation for successful application with differing serotypes. Though caveats apply for anomalous materials which may co-elute with the main peak, we find this method highly suitable for routine empty:full ratio determinations, and highly advantageous given the additional information (mass confirmation, size check, level of aggregates) collected simultaneously.
Darren Begley is a senior scientist working in the Analytical Development (AD) group at Beam Therapeutics. Beam is pioneering the use of base editors to develop a new class of precision genetic medicines. Darren has helped build and grow the AD team, providing biophysical testing capabilities both internally and through strategic external collaborators. for a variety of materials, including small and large oligonucleotides, protein-nucleic acid complexes, lipid nanoparticles (LNPs), and adeno-associated virus (AAV) particles. Prior to joining Beam, Darren worked with AAV and virus-like particles at Ultragenyx and Wolfe Laboratories. He obtained a PhD in chemistry with a structural biology focus on RNA and small molecules at the University of Washington in Seattle, and has a Bachelor of Science degree from McGill University in Montréal.
Development and Optimisation of Virus-Like Particle Vaccines Against Pathogenic Arboviruses
Chikungunya virus and Mayaro virus are emerging pathogens that cause debilitating arthritic disease in humans, whereas Zika virus is associated with brain malformations in the unborn child. These viruses are transmitted to humans by mosquitoes, and no licensed vaccines or antiviral drugs are currently available for human use. We developed enveloped virus-like particle (VLP) vaccines against chikungunya virus, Mayaro virus, and Zika virus using the scalable baculovirus-insect cell expression system. Moreover, we aimed to increase VLP production yield using methods that inhibit RNA interference in insect cells and enhance VLP budding from insect cells. High-level secretion of VLPs into the culture fluid of insect cells was achieved at volumes reaching bioreactor-scale, and particles with correct diameters were observed after purification. Challenge experiments to assess the ability of the different VLP vaccines to confer protection in mice are currently ongoing.
Sandra Abbo is a PhD candidate at the Laboratory of Virology at Wageningen University, the Netherlands. Within the ZikaRisk project she investigates the vector competence of mosquitoes from the Netherlands for Zika virus, and she tries to understand the molecular mechanisms underlying the vector specificity of Zika virus. Moreover, she also works on the development and optimisation of VLP vaccines against multiple pathogenic arboviruses. Sandra obtained her MSc degree in biotechnology cum laude at Wageningen University in 2016. During her MSc internship, she worked on cancer cell metabolism at the MRC Cancer Unit of the University of Cambridge, United Kingdom. She then continued with her MSc thesis research about VLP vaccine development at Wageningen University, where she received the Van der Want award for the best MSc thesis in Virology.
Associate Professor of Biochemistry & Molecular Biology
Ongoing Work for Expression of Difficult-to-Express Proteins and Protein Complexes
Development of recombinant protein expression technologies has been one of the cornerstones in modern molecular biology. Recombinant protein expression is based on cloning genes encoding the protein of interest into the vector(s) followed by delivering the cloned genes into the host cells for heterologous protein expression. Despite an array of recombinant protein expression systems, one fundamental problem remains practically unsolved — to express large and often problematic proteins in a reasonable quantity in soluble form in a consistent fashion. We developed a new technology that enables us to express large and often problematic proteins in insect cells. Our system uses a synthetic artificial protein that enhances the solubility of proteins, thereby enabling expression of large and often problematic proteins.
Yuichiro Takagi, PhD is Associate Professor at the Indiana University School of Medicine. He received a BS degree from Ibaraki University, an MS degree from the University of Tokyo, and his PhD in biochemistry from the University of Oklahoma. He conducted postdoctoral research in the laboratory of Dr. Roger Kornberg at Stanford University. The Takagi laboratory is interested in understanding the mechanisms of assembly, structure, and function of large multi-protein complexes involved in eukaryotic gene regulation. His lab is particularly interested in the development of new expression technologies which enable production of large and often problematic proteins and protein complexes for structural (x-ray crystallography and cryo-EM) and functional studies.
Development and Validation of a Potency Assay for a Viral-Based Gene Therapy Product
Viral-based gene therapy is relatively new and gaining momentum with promising early successes in clinical and regulatory approval. The potency assay is typically the most customized and unique among the battery of product release and stability indicating assays. Unlike other biological drugs, the potency assay for a gene therapy product involves multiple steps including infectivity, transcription, translation, protein modifications, localization of the protein product, and protein function. The potency assay for a gene therapy product is extremely challenging, especially for AAV viral vectors, as it has poor infectivity and limited sensitivity. A successful potency method must evaluate infection, transcription, and function of the resulting protein. Here we present our approaches and strategies used in the development and validation of a potency assay for a viral-based gene therapy product.
Rashmi Prasad, PhD is an experienced scientist with a demonstrated history of working in the biotechnology industry. She is skilled in protein analytical biochemistry, protein purification, and molecular biology tools. Dr. Prasad works as Scientist II in the Quality Control team at MassBiologics in Massachusetts. Her major focus area is the qualification and validation of bioanalytical assays for viral vectors (AAV) targeted to characterize the products and in-process samples of gene therapy. She also supports her team as the Technical SME of major bioassays. Rashmi is a strong research professional who earned her PhD in Biochemistry for work on yeast chromatin remodeling enzymes and their crosstalk with histone chaperones, from Southern Illinois University in Carbondale.
Building Effective Control Strategies for Cellular Therapy and Other Complex Biopharmaceutical Products
As biopharmaceutical products become more complex, the methods for developing and validating them must become more sophisticated. Today’s products, especially those for cell and gene therapies, have a large number of both known and unknown critical quality attributes (CQAs/u CQAs) that must be controlled over their entire development and manufacturing lifecycle to assure both successful development and sustained, reliable manufacturing of high-quality product. The talk covers recently developed methods that include lifecycle process development and validation (LPDV) using working-quality by design (w QbD) and a well-structured design space (ws DS) for systematically building process control strategies for both CQAs and u CQAs. The talk also covers recently developed quality risk management (QRM) methods that provide mechanisms for rapidly identifying and screening the large number of process parameters to speed process development and provide for more effective use of design of experiments (DOE) methods. When used in combination, these methods provide more straightforward approaches to rapidly developing and communicating well-defined processes for commercializing 21st century products.
Mark has over 35 years of experience designing and operating biopharmaceutical manufacturing facilities. Recently he has been consulting for companies on product, process, and manufacturing capacity development for new products. He was a member of NNE’s Strategic Manufacturing Concept Group and previously with IPS working on feasibility and conceptual design studies for advanced biopharmaceutical manufacturing facilities. Before working for engineering companies, Mark, while helping raise his two kids, was an independent consultant in the biopharmaceutical industry for 15 years on operational issues related to: process, product, and strategic business development; clinical and commercial manufacturing planning; tech transfer; and facility design and construction. For many years, he taught an ISPE Biopharmaceutical Process Validation course. He was previously the Sr. Vice President, Manufacturing Operations for the CMO Covance Biotechnology Services (formerly Corning Bio, Inc.). Prior to joining Covance, he was Vice President of Manufacturing at Amgen, Inc. Mark was with Amgen for nine years and held positions as Engineering Manager, Plant Manager, and Director of EPOGEN® Manufacturing. Mark received his PhD in Chemical Engineering from the University of Massachusetts.
Machine Learning Applications for the Characterization of Particle Profiles of Therapeutic Products
Flow imaging is a proven method for characterization of particulates in therapeutic products. It is routinely performed alongside the USP 788/787 Light Obscuration methods to more accurately quantify and characterize the particle subpopulations in drug products (silicone oil, protein aggregate, extrinsic material, etc.). Typical classifications of imaging data use single parameter filters such as aspect ratio to quantify silicone oil compared to protein. However, machine learning provides a sophisticated approach to more accurately classify particles in therapeutic products by leveraging the information present in the raw particle images. We will demonstrate how various machine learning algorithms facilitate improved classification compared to the traditional approach, leading to superior sample descriptions. We provide examples of the benefits that machine learning provides for cell therapy products. Flow imaging has tremendous potential to monitor particle size distributions, aggregates/agglomerates, and extrinsic contaminants from batch to batch. Applying machine learning to flow imaging of pharmaceutical products can assist in defining the criticality of product quality attributes, as well as establishing an integrated control strategy for characterization and control of drug products.
Amber currently holds the position of Director at KBI Biopharma where she manages the Particle Characterization Core that specializes in analytical methods for quantifying, characterizing, and identifying particulates. She received her PhD in Chemical Engineering within the Pharmaceutical Biotechnology Program with a specialization in the field of protein stability at the University of Colorado at Boulder. Previously, at Amgen, Amber was a Scientist within the biomolecular structures and interactions group where she supported biophysical characterization of protein products with a specialty in subvisible particle characterization and identification. She has over 12 years of experience with analytical method development and validation, formulation strategy, and protein biophysical characterization.
Automation in Delivery Technologies to Empower Breakthrough Therapies
With the advent of personalized medicines, the ability to deliver payloads in a precise and efficient manner into cells becomes more integral to the success of the therapy. Although the existing strategies of viral vectors and electroporation allow for mass manipulation of cells, they lack the ideal precision to be used on fragile cell types for certain applications. Thus, many emerging technologies are focusing on strategies to improve accuracy by navigating delivery at the single cell level. The goal of any delivery technology is to create a system of automation that can be easily scalable and translatable to manufacturing of these new therapies. We will discuss the strategies behind creating a platform that can be universally adapted and automated for a variety of applications that can make manufacturing of personalized therapies much more cost-effective and efficient.
Dr. Anil Narasimha is the co-founder and CEO of Mekonos, a San Francisco, California-based company developing a platform for universal delivery of payloads into cells. After receiving his BS from the University of California, Berkeley, he completed his PhD at the University of California, San Diego. He then completed a postdoctoral fellowship at Stanford University in Dr. Michael Snyder’s group, where he co-founded Mekonos.
Advanced Therapies, Clinical Holds, and Strategies to Avoid
The global landscape for cell therapies is robust with a reported 1.6 billion dollars in financing and more than 200 ongoing clinical trials as of the third quarter of 2019. These cell therapies include allogeneic, autologous, and primary cells. The global landscape of approved therapies includes six cellular immunotherapies and 20 cell therapies. The number of clinical trials that rely on cells is rapidly evolving from those isolated from perinatal sources to those relying on mesenchymal/stromal cells. Many of these products are being developed under expedited approval designations such as the US FDA Regenerative Medicine Advanced Therapy (RMAT), European Medicines Agency (EMA) PRIME, or Japan’s Sakigake. Recently clinical holds have been reported for several advanced therapies under clinical investigation under an approved investigational new drug application (IND). The reasons for clinical hold have included adverse events, delivery device used, and manufacturing. What constitutes a clinical hold is specifically codified in the US Code of Federal Regulations under 21 CFR 312.42. Of these, insufficient information and unreasonable risk are particularly relevant with respect to manufacturing, as problems with manufacture can convey a risk leading to a clinical hold. This is particularly important for cellular therapies where quality, safety, and efficacy are all intertwined. FDA can issue a clinical hold for an initial IND via the issuance of a complete response letter or place an ongoing clinical trial on hold by issuing a clinical hold letter. While news regarding clinical holds of ongoing investigations is typically public information, there is less visibility with respect to holds placed on initial INDs. Three publications by FDA staff have examined the reasons for INDs placed on clinical holds. CMC or quality issues have ranked first or second as the primary reason cited for the clinical hold and submitting with incomplete information was the cited reason in greater than 90% of all clinical holds. Important strategies for avoiding a clinical hold include beginning with the end in mind, maintaining alignment among quality, nonclinical, and clinical stakeholders, maintaining data integrity, and accurate, clear communication.
Debra Aub Webster, PhD has over 20 years of experience in pharmaceutical research and the regulatory environment. She received her undergraduate degree from Virginia Polytechnic Institute and State University, and her graduate degree in pharmacology and toxicology from the Virginia Commonwealth University’s Medical College of Virginia. After leaving academia and bench research, Dr. Webster joined the US Food and Drug Administration (FDA) as a reviewing toxicologist. Dr. Webster was an inaugural member of the FDA’s Division of Anti-Viral Drugs in the Center for Drug Evaluation and Research. Here she was responsible for critical evaluation of the nonclinical pharmacology and toxicology sections of investigational new drug applications (INDs) and new drug applications (NDAs). She authored reviews, advisory opinions, and executive summaries, and was awarded a Medal of Appreciation from the Commissioner for her work. Prior to leaving the FDA, she held a rotation as Assistant to the Director for Pharmacology/Toxicology. Dr. Webster is the Director of Advanced Therapy Product Development at Cardinal Health Specialty Solutions. She is responsible for providing regulatory strategy consulting support across clinical, nonclinical, and CMC to lead product development from proof-of-concept through to approval. Dr. Webster has provided support and leadership for the development of regenerative medicines and advanced therapies, including CAR T-cell and other genetically engineered cells, stem cell therapies, gene therapies, genome editing therapies, oncolytic virus therapies, therapeutic cancer vaccines, recombinant human proteins, antibody-based therapies, biosimilars, and bioengineered tissue constructs.
Considerations for Plasmid DNA in Gene Therapy: Evaluating the Impact of How Materials are Used on Design, Characterization, and Specifications
Recent advances in gene therapy have made treatments based on reprogramming cells to treat disease a reality. Viral vectors are addressing monogenic defects with protein replacements, delivering therapeutic proteins, and reprogramming T-cells to target cancer. Each of these approaches needs a variety of nucleic acids and enzymes to achieve the therapeutic benefit. How each of these are used, their proximity to the patient, the route of administration, and other factors impact the requirements of these materials. We will review examples of how raw materials are used in these therapies and how best to evaluate the design, characterization, and specifications in the context of their use. We will present some of the key requirements that drive cost and timeline with a goal to optimize production and ensure the safety and efficacy of the product. We will discuss the spectrum of needs that must be addressed, from off-the-shelf products to enable large-scale production of a single therapy to “off-the-shelf production methods” to enable small-scale production of many individual therapies.
Michelle Berg brings over 20 years of experience in the biotechnology sector, ten years of which have been spent in executive leadership roles. In her current position as President of Aldevron’s GMP Nucleic Acids Business Unit, Berg oversees Aldevron’s strategy to provide GMP plasmids and mRNA for gene editing, gene therapy, and cell therapy applications. She works closely with the company’s operational team to meet the requirements of these growing fields, while supporting the clinical and commercial efforts of Aldevron’s clients. Aldevron’s GMP plasmid facility, located in Fargo, North Dakota, is the largest in the world. Berg earned a Bachelor of Science in Biotechnology from North Dakota State University (NDSU). She is a contributing author and speaker on patient-focused programming, rare disease advocacy, and accessible education on genetic medicines. In addition to her roles with Abeona and Aldevron, she has performed research on behalf of the Department of Plant Sciences at NDSU.
Enhancing Cryopreservation Outcome through Molecular and Device-Based Strategies
The discipline of cryopreservation (CP) covers a wide diversity of application areas ranging from cell line banking to cell therapy. While serving a critical role, protocols, approaches, and technologies have evolved little over the last several decades. This has resulted in a bottleneck impacting numerous areas including cell therapy, tissue engineering, and tissue banking. With established techniques, CP is often viewed as an “old school” discipline; yet modern CP is in the midst of another scientific and technology development growth phase as CP finds itself on the frontlines of molecular biology, bioengineering, and clinical medicine. In this regard, numerous studies have described the impact, mechanisms, and points of control of cryopreservation-induced delayed-onset cell death (CIDOCD). Despite this well accepted phenomena, today most researchers still rely on traditional CP methodologies, assessment of cell survival immediately post-thaw (within a few hours), and fail to account for the impact of CIDOCD which continues to compromise survival hours to days post-thaw. In an effort to limit CIDOCD, researchers are now focused on developing new CP agents, freeze media formulation devices, etc. to provide a path to improve survival compared to traditional media plus DMSO approaches. Despite reported improvements, many of these developments have yet to enter mainstream utilization. While the ultimate end use may vary significantly, the overall objectives of CP remain the same: to obtain the highest quality cell product (survival and function) following thawing. This presentation will provide an overview of our current understanding of the molecular stress response of cells to cryopreservation, the interrelated role of the apoptotic and necrotic cell death continuum, and how this impacts outcome. Discussion will cover the “dos and don’ts of cryopreservation today” with a focus on the impact of molecular control, buffering of cell stress response during and following CP, and how cryopreservation media (intracellular vs. extracellular), cryoprotective agents (DMSO concentration, etc.), storage conditions, warming/thawing (protocols and devices), recovery media (normal culture vs. stress control) and viability assessment (immediate vs. 24 hours) impacts outcome. Data presented will include cell viability and molecular stress results from various cell systems including several cell lines as well as human stem cells (hHSCs, hMSCs). The data will illustrate how the utilization of new approaches at each stage enables more efficient and reliable cryopreservation thereby yielding increased sample viability and functionality in comparison to traditional methodologies. Importantly, these improvements are now providing for a new foundation to accelerate new research, technology, and product development for which CP serves as an integral component.
John M. Baust, PhD is the Founder, President, and Lead Scientist of CPSI Biotech. Dr. Baust has over 20 years’ experience in research and medical device development and is a co-inventor on over 75 patents. Dr. Baust is a recognized innovator and entrepreneur in cryomedicine and is a pioneer in the area of molecular mechanisms of cell death and low temperature stress. Dr. Baust has published over 100 papers, reviews, book chapters, abstracts, and patents in the area of low temperature biology and has been instrumental in the advancement of the field of cryobiology into the molecular biological era focusing on signal transduction and apoptosis. In this regard, Dr. Baust is credited with the discovery of cryopreservation-induced delayed-onset cell death. In the area of research and technology development, Dr. Baust has led the development of numerous medical devices, including the supercritical nitrogen (SCN) and pressurized nitrogen (PSN) cryoablation devices for the treatment of cancer and cardiac arrhythmias. This is in addition to spearheading the development of the SmartThaw device for improved cell and tissue cryopreservation. Coupled with these technical engineering developments, he leads life science research programs focused on the cell-molecular actions of cryoablation. These efforts have resulted in the identification of a significant molecular stress response component to freezing injury which is responsible for the differential sensitivity of various cancers to thermal ablation. As a entrepreneur, Dr. Baust has founded or been on the founding team of five biotech and medtech companies. He has been involved with the development and commercialization of several products including CryoStor and HTS-FRS (BioLife Solutions, Inc). In addition to these activities, Dr. Baust serves on the editorial boards of Biopreservation and Biobanking as well as Technology in Cancer Research and Treatment, and is a reviewer for several other scientific journals. Dr. Baust co-edited the book Advances in Biopreservation, and is a past board member for the Society for Cryobiology and the American College of Cryosurgery. Dr. Baust completed his studies at Cornell University, State University of New York at Binghamton, and Harvard Medical School.
Optimization of Adeno-Associated Virus Production in a Chemically-Defined Sf9/Baculovirus System
Adeno-associated virus (AAV) is among the most commonly-used viral vectors, currently representing ~23% of the gene therapy market. To date, there have been three approved AAV products for the treatment of disease, and there are currently greater than 300 development projects and clinical trials ongoing worldwide. A roadblock faced by researchers and clinicians is the production of suitable amounts of viral vectors for their preclinical and clinical trials, as well as for patient treatment. Production of AAV in insect cells is poised to address material demand, as AAV production runs at the 2000 L bioreactor-scale have been reported with overall volumetric yields in the range of 1016–1017 genomic (VG) particles. Here we present data showing the production of AAV in a chemically-defined Sf9/baculovirus system. Data will be presented on AAV production optimization and purification.
Kenneth Thompson, PhD is a Manager of Cell Biology R&D in the Life Sciences Solutions Group at Thermo Fisher Scientific in Frederick, Maryland. Dr. Thompson leads a team of scientists focused on developing new products for cell biology applications including baculovirus-based insect systems for protein expression and virus production. Dr. Thompson received his PhD in Molecular and Cellular Biology and BS in Biological Sciences from the University of Maryland, Baltimore County.
Vaccine and Viral Vector Production Using the Baculovirus-Insect Cell Expression System: AAV Case Study
The insect cell-baculovirus expression system (IC-BEVS) has been extensively used as a platform for production of vaccines and viral vectors for gene therapy applications. Our lab has dedicated significant efforts to improve the production of influenza-like virus particles as a potential influenza vaccine candidate. In this regard, major manufacturing challenges have been identified including the difficulty to separate baculovirus from VLPs in the final formulation. Furthermore, we have explored overcoming bottlenecks known to affect vaccine production in the insect cell platform by using RNAi-mediated silencing. A critical review and understanding on the topic guides us in future directions using this approach. On the other hand, our research work has also been focused on the optimization of the production process of adeno-associated viral vectors (AAV) in the IC-BEVS system. Recently, the implementation of an efficient production process using the OneBac system to produce AAV5 has been achieved. The volumetric yields of AAV genomic particles were significantly increased by applying a refined fed-batch strategy achieving high cell densities in bioreactors. Additionally, substantial improvements to the downstream processing of the AAVs and separation of empty and full capsids at preparative-scale has been attained. These findings support the feasibility and promise of the IC-BEVS system as a remarkable platform to support the industrial manufacturing of biological products.
Chief Technology Officer and Manager of Protein Purification
BacMam-Mediated In Situ Modification of Recombinant Proteins
Viral transduction is one of the most efficient mechanisms for the introduction of foreign genes into mammalian cells. Advantages associated with this method of gene transfer include ease of gene amplification, reproducible efficiencies of gene transfer and the ability to tightly regulate the number of gene copies introduced into the host cell. This latter characteristic is of particular interest in the formation of protein complexes such as VLPs and the ability to successfully produce difficult to express proteins like equine encephalitis E1 and E2 antigens in soluble form. BacMam viruses are BSL1 entities that have the ability to carry large inserts (at least 15 kb) and are extremely easy to amplify and use. A potential use of the BacMam technology that we have been working with is its use to drive modifications of overexpressed proteins in situ. In this presentation we describe the development of a BacMam viral construct carrying the gene for BirA to catalyze the biotinylation of rIgG produced in HEK-293 cells transfected using PEI. In a second application a BacMam virus with the gene for human furin inserted was used to overexpress furin in HEK-293 cells transfected with genes carrying furin cleavage sites. Data related to the development of the two processes and their functionality will be presented.
Production of AAV Vectors: Improvements and Innovations
Otto-Wilhelm Merten has a PhD in biotechnology from the University of Natural Resources and Life Sciences in Vienna, Austria (Institute of Applied Microbiology, Prof. H. Katinger), holds an HDR at the University of Évry Val d'Essonne, France, and is Visiting Professor at ITQB/UNL in Lisbon, Portugal. His scientific activities brought him from the Sandoz Forschungsinstitut in Vienna to the Institut Pasteur in Paris, to Généthon in Evry, France, and more recently to Lentigen/Miltenyi Biotec in the US. As a biotechnologist he was/is involved in cell culture development and optimisation of production, purification, and analytical methods for recombinant proteins (to a large extent monoclonal antibodies), viruses, including rabies and influenza virus, and viral vectors (murine leukemia virus [MLV], lentivirus [LV], and adeno-associated virus [AAV]). With respect to LV and AAV vectors he was involved in the development of large-scale production methods for their routine manufacturing. In context of his expertise in animal cell culture technology he was a long-term member of the Executive Committee of the European Society of Animal Cell Technology (ESACT) and served as its chairman from 2001 to 2005. Today he is an honorary member of ESACT, as well as a member of scientific societies in the field of gene and cell therapy (ESGCT, ASGCT). He is also a member of the scientific editorial board of Human Gene Therapy and of Molecular Therapy - Methods & Clinical Development.
Principal Investigator/Supervisory Microbiologist, Office of Vaccines Research and Review
Dr. Arifa S. Khan received her PhD in Microbiology from the George Washington University, Washington, DC. She is currently a Supervisory Microbiologist and Principal Investigator in the Division of Viral Products, Office of Vaccines Research and Review in the Center for Biologics Evaluation and Research (CBER), US Food and Drug Administration (FDA). Dr. Khan joined the FDA in 1991 after working in Dr. Malcolm Martin’s laboratory at the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) since 1979, where she contributed significantly to the field of murine leukemia retroviruses, endogenous retroviruses, and simian immunodeficiency virus (SIV). In CBER, Dr. Khan established and maintains a rigorous research program on the development of sensitive, state-of-the art assays for adventitious virus detection, with a focus on safety of novel cell substrates and vaccines. Dr. Khan’s current research efforts include standards and standardization of next-generation sequencing for adventitious virus detection in biologics. Her regulatory responsibilities include review of candidate viral vaccines for HIV and emerging viruses such as SARS-CoV-2, and to provide expert consultation on novel cell substrates and adventitious viruses in CBER and CDER. Dr. Khan has been involved in licensure of several viral vaccines and development of FDA, International Conference on Harmonisation (ICH), Public Health Service (PHS), United States Pharmacopeia (USP), and World Health Organization (WHO) guidance documents related to cell substrates and viral safety. She is the FDA lead on adventitious viruses and next-generation sequencing for cell substrates and product safety.
Senior Vice President of Manufacturing
New Nanotechnology for Efficient Non-Viral Gene Delivery to Cells Ex Vivo
Dr. Ballas has over 20 years’ experience working on cell and gene therapies in academic and industry roles with expertise in manufacturing of viral vectors and cell therapies. He served as Vice President of Manufacturing for publicly traded Rocket Pharmaceuticals, and prior to that as Senior Director of Process Development and Commercialization for publicly traded WuXi AppTec where he was responsible for manufacturing operations and program management for gene therapy clients. Previously he ran autologous cell therapy clinical trials for Cook Medical, and held key positions for cell and gene therapy core programs at Indiana University School of Medicine. He co-developed a ultra-high throughput microinjection device (US9885059) and co-founded Basilard Biotech to commercialize this technology. Chris holds a PhD in cellular and molecular pathology from Vanderbilt University School of Medicine and was a Children’s Brittle Bone Foundation fellow during a postdoctoral fellowship at Case Western Reserve University School of Medicine.
Development and Validation of Analytical Methods for GTx Commercialization
PhD Candidate, Biological & Biomedical Engineering, Viral Vectors and Vaccine Bioprocessing Group